Prevention of cell migration initiation with CMV US28 receptor antagonists

ABSTRACT

There is disclosed an assay system for determining therapeutic activity for treating restenosis, atherosclerosis, chronic rejection syndrome and graft versus host disease (GVHD) by measuring inhibition of cell migration activity in smooth muscle cells expressing a US28 receptor from the CMV genome. Specifically, there is disclosed a method for measuring inhibition of cell migration in isolated cells transfected with US28 or infected with CMV and stimulated with a ligand. There is further disclosed a method for treating atherosclerosis, restenosis, chronic rejection syndrome and graft versus host disease (GVHD), comprising administering an effective amount of an agent that is a US28 receptor antagonist, wherein a US28 receptor antagonist comprises an inhibitor compound that prevents transduction of US28 receptor signal stimulated by a US28 receptor ligand, wherein a US28 receptor ligand is selected from the group consisting of RANTES, MIP-1α and MCP. The invention further provides a method for treating restenosis, atherosclerosis, chronic rejection syndrome and GVHD by administering KHSV encoded vMIP-2, fractalkine or herbimycin.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention provides an assay system for determiningtherapeutic activity for treating restenosis, atherosclerosis, chronicrejection syndrome and graft versus host disease (GVHD) by measuringinhibition of cell migration activity in smooth muscle cells expressinga US28 receptor from the CMV genome. Specifically, the present inventionprovides a method for measuring inhibition of cell migration in isolatedcells transfected with US28 or infected with CMV and stimulated with aligand. The invention further provides a method for treating restenosis,atherosclerosis, chronic rejection syndrome and GVHD by administeringKHSV encoded vMIP-2, fractalkine or herbimycin.

BACKGROUND OF THE INVENTION

[0002] Atherosclerosis, Restenosis, Chronic Rejection Syndrome and GVHD

[0003] Atherosclerosis is a major cause of morbidity in theindustrialized world. Atherosclerotic lesions usually become apparent inadult patients as a result of complete occlusion of a strategic bloodvessel and the resulting complication. However, such lesions begin muchearlier in the life of the patient. It was later noticed that there wasa statistical association with viral infection, particularly CMV.

[0004] It has been postulated that CMV and possibly herpes virus areinvolved in the inducement of atherosclerotic lesions. Severalinvestigators have demonstrated the presence of CMV nucleic acids and/orantigens in the human arterial wall using DNA hybridization techniques(Melnick et al., Lancet 11:644-647, 1983), immunohistochemistry (Petrieet al., J. Infect. Dis. 155:158-159, 1987), dot blot and in situhybridization techniques (Hendrix et al., Am. J Path. 134:1151-1157,1989), and by polymerase chain reaction (PCR) techniques using probesderived from immediate early and late genomic regions (Hendrix et al.,Am. J Path. 136:23-28, 1990). Thus, there has been finding of viralantigens and nucleic acid sequences in arterial smooth muscle cells thatsuggest that CMV infection of the arterial wall may be a commonoccurrence in patients with atherosclerosis.

[0005] Soon after renal transplantation became an accepted treatment, anassociation was noted between CMV infection, glomerulopathy, andrejection of the transplanted kidney. Thus, CMV was investigated todetermine if it played a role in graft atherosclerosis that frequentlyoccurs after heart transplantation (Grattan et al., J. Am. Med. Assn.261:3561-3566, 1989). The findings show that heart transplant patientswho are immunosuppressed and become infected with CMV are particularlyprone to develop atherosclerosis in the transplanted organ. It ispostulated that the artery wall may be the site of CMV latency becauseCMV DNA but not infectious virus was found in the artery wall.

[0006] Role of Chemokines

[0007] Chemokines are chemoattratants for neutrophils, monocytes,lymphocytes and bone marrow progenitors, as well as other cell types.The family of chemokines comprises four subfamilies, defined by thedistribution of cysteine residues in the N terminus of these factors,the CXC, CC, C, and CX3C subfamilies. The chemokines are related byprimary structure, particularly by conservation of a four-cysteinemotif. C-C chemokines include such members as human monocyte chemotacticprotein 1 (MCP-1), RANTES, and the macrophage inflammatory proteins 1αand 1β (MIP-1α and MIP-1β). These ligands exhibit chemoattractantpotential for monocytes but not neutrophils. CMV infection can alsomodify the level of chemokines. The level of RANTES (a chemokine)produced by cells recovered by bronchoalveolar lavage from lungtransplant patients with CMV pneumonitis shows that cells from infectedpatients secreted greater amounts of RANTES than did cells recoveredfrom either patients undergoing acute rejection or from control subjects(Monti et al., Transplantation 61:1757-1762, 1996). AIDS patients withCMV encephlitis have higher concentrations of MCP-1 (a chemokine) butnot other chemokines in their spinal fluid than do HIV seropositivepersons who are asymptomatic or AIDS patients with a number of otheropportunistic infections of the central nervous system (Bernasconi etal., J. Infec. Dis. 174:1098-1101, 1996). When fibroblasts were infectedwith CMV, RANTES mRNA and protein expression are induced early, butextracellular RANTES accumulation, but not transcription isdown-regulated late during CMV infection (Michelson et al., J Virol.71:6495-6500, 1997). Therefore, CMV infection has the capacity to bothinduce cell migration and enhance chemokine production early during theinfection process.

[0008] Chemokine receptors tend to be multiple membrane-spanningproteins, generally 7 or 8 membrane-spanning proteins and tend totransduce signal through G-coupled protein signal transduction. HumanC-C chemokines tend to bind to the US28 receptor of CMV (Neote et al.,Cell 72:415-425, 1993). There is also a sequence homology between theC-CKR-1 receptor (normal human gene) and the CMV US28 sequence in theopen reading frame region. (Neote et al., 1993). Thus, Neote et al.speculated that “the protein encoded by the US28 open reading frame ofTowne strain CMV can bind C-C chemokines but not the C-X-C chemokineIL-8. However, none of the earlier chemokine receptor papers, includingNeote et al., has made the connection between US28 and it s role inmediating smooth muscle cell proliferation.

SUMMARY OF THE INVENTION

[0009] The present invention provides an assay for determiningtherapeutic activity of US28 receptor antagonists, comprising (a)obtaining and isolating smooth muscle cells into a first chamber of amigration device, wherein the first migration chamber comprises growthmedia chambers and is defined by a first side of a membrane and chamberwalls, and wherein the migration device comprises a second chamberdefined by the second side of the membrane and having an enclosed space;(b) infecting the smooth muscle cells with human cytomegalovirus (HCVM)containing a gene encoding the US28 receptor; (c) adding a candidatetherapeutic agent to the first chamber; and (d) determining the amountof cellular migration into the second chamber, whereby inhibition ofcellular migration of infected smooth muscle cells indicates therapeuticactivity. Preferably, the smooth muscle cells are isolated frompulmonary arteries. Preferably, the membrane has a pore size of fromabout 2 to about 10 microns. Most preferably, the membrane pore size isabout 3 microns. Preferably the amount of cellular migration isdetermined by an assay for counting the number of smooth muscle cells inthe second chamber wherein the assay for counting the number of smoothmuscle cells is selected from the group consisting of microscopic cellcounting per unit area, radiolabeling the smooth muscle cells andcounting radioactivity in the second chamber, attaching a fluorescentprobe to the smooth muscle cells and measuring fluorescence within thesecond chamber, and combinations thereof.

[0010] The present invention further provides a method for treatingatherosclerosis, restenosis, chronic rejection syndrome and graft versushost disease (GVHD), comprising administering an effective amount of anagent that is a US28 receptor antagonist, wherein a US28 receptorantagonist comprises an inhibitor compound that prevents transduction ofUS28 receptor signal stimulated by a US28 receptor ligand, wherein aUS28 receptor ligand is selected from the group consisting of RANTES,MIP-1α and MCP. Preferably, the US28 receptor antagonist is selectedfrom the group consisting of an antibody that binds to an extracellularportion of the US28 receptor, and an antisense oligonucleotide having anucleic acid sequence antisense to the US28 cDNA and inhibitingtranslation of US28 expression in infected smooth muscle cells, or aUS28 binding antagonist, wherein the US28 binding antagonist is selectedfrom the group consisting of KHSV encoded vMIP-2, fractalkine, andherbimycin. Preferably, the monoclonal antibody is chimeric or humanizedby means for humanizing non-human antibodies. Preferably, the US28antisense sequences are selected from the group consisting of SEQ IDNOS. 2-28.

[0011] The present invention further provides a method for enhancingcellular migration, comprising infecting a cell with a viral nucleicacid containing a gene encoding CVM US28 receptor or tansfecting a cellwith a vector comprising the cDNA sequence for US28 operably linked to aviral promoter sequence, and stimulating the transfected or infectedcell with a US28 receptor ligand, selected from the group consisting ofRANTES, MIP-1α and MCP1.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows the ability of HCMV-GFP (human cytomegalovirus GFP)to infect pulmonary smooth muscle cells (SMC) in vitro. SMC wereinfected with HCMV-GFP (MOI 10) for 2 days and then examined for thepresence of GFOHCMV immediate early expression. Colocalization of GFPand IE (intermediate-early) were observed only in HCMV-GFP infectedcells.

[0013]FIG. 2 shows that HCMV infected SMCs were examined by electronmicroscopy for the presence of virus. The photos show numerous viruscapsids were found in the nucleus and mature virions were observed onthe plasma membrane of HCMV infected cells.

[0014]FIG. 3 shows a one-step growth curve showing HCMV replication inSMCs. SMC were infected with HCMV Towne strain at MOI1. These data showHCMV growth and release in SMC exhibited normal kinetics.

[0015]FIG. 4 shows that HCMV infection induced actin reorganization inSMC as an indication of migration activity. The cells were treated witheither PDGF (100 ng/ml) of infected with HCMV Towne strain at MOI1.Actin distribution was visualized at 5 days post-treatment byfluorescence using TRITC conjugated phalloidin.

[0016]FIG. 5 shows an SMC migration assay scheme. SMCs are cultured inthe upper chamber and infected with HCMV (preferably at or near MOI1).Only cells that are infected will migrate through a filter (preferably 3micron) to the lower chamber. The cells in the lower chamber are countedby microscopy or labeled with a radioactive or fluorescent label.

[0017]FIG. 6 shows the presence of HCMV in migrating SMCs. SMC wereinfected with HCMV-GFP. The migrating cells were analyzed for thepresence of GFP and HCMV glycoprotein gB by immunofluorescence usinganti-gB antibodies. All of the migrating SMCs exhibited GFP and gBexpression.

[0018]FIG. 7 shows that HCMV induced migration of SMCs. Cellularmobility assays were used to determine the specificity of HCMV inducedSMC migration. SMCs that were treated with PDGF (Platelet-derived growthfactor, an inducer of cellular migration) did not cause cell migrationin SMCs to nearly the extent as HCMV infection. Moreover, HCMVneutralizing antibodies reduced cellular migration to mock levels.

[0019]FIG. 8 shows that HCMV-induced migration was specific for SMCs.Both SMCs and human foreskin fibroblasts (HFF) were infected andanalyzed in migration assays. The data provided in FIG. 8 show that HCMVinduced cellular migration occurred only in SMCs but not insimilarly-infected HFFs.

[0020]FIG. 9 shows that protein synthesis was required for HCMV-inducedSMC migration. SMC mobility was blocked by cyclohexamide (a generalprotein synthesis inhibitor). Moreover, foscarnet (an inhibitor of HCMVlate gene production) did not inhibit SMC migration.

[0021]FIG. 10 shows that HCMV genome encodes four putative chemokinereceptors, including US27, US28, UL33 and UL78.

[0022]FIG. 11 shows that HCMV infection of HFFs induced RANTES(chemokine) expression. HFF cell culture supernatants were collectedevery 8 hours from infected HFFs and RANTES concentrations in thesupernatants were determined by an ELISA assay.

[0023]FIG. 12 shows that the addition of RANTES at the concentrationsshown to HCMV-GFP-infected SMCs increased SMC migration in adose-dependent manner.

[0024]FIG. 13 shows that that a HCMV having the US28 receptor genedeleted affected cell motility.

[0025]FIG. 14 shows the construction scheme for human CMV GFPrecombinants.

[0026]FIG. 15 shows the results of inhibition of a PTK pathway effect ofUS28 SMC migration with several PTK inhibitors including herimycin A,pertussis toxin and genistein at the concentrations indicated. Pertussistoxin had no effect.

DETAILED DESCRIPTION OF THE INVENTION

[0027] US28 Receptor

[0028] The sequence characterization of the US28 receptor is provided inNeote et al. (Cell 72:415-425, 1993) and also the cDNA sequence is SEQID No. 1. The present invention is based upon the discovery that the CMVeffect in causing smooth muscle proliferation and an initiating event inthe diseases atherosclerosis, restenosis, chronic organ rejection andGVHD, is mediated primarily through signal transduction in infectedsmooth muscle cells through the US28 receptor. Based upon thisdiscovery, described herein, the claimed invention is provided thatprovides US28 receptor antagonist molecules that have therapeuticeffect. Moreover, the present invention provides an assay procedure toscreen of other US28 antagonist molecules that, based upon the findingsreported here, are effective for treating atherosclerosis, restenosis,chronic organ rejection and GVHD.

[0029] Role of US28 in Smooth Muscle Cell Migration

[0030] The present invention is based upon the discovery of the role ofUS28 in mediating the properties of CMV to stimulate smooth muscle cellsthat can ultimately lead to atherosclerosis, restenosis, chronicrejection syndrome or GVHD in susceptible patients. It is clear to askill practitioner that a patient must first have been a transplantrecipient before he or she is at risk for either GVHD or chronicrejection syndrome. Moreover, restenosis first requires anangioplasty-type procedure or other chemical or surgical intervention inclearing occluded or partially occluded arteries before the patient isat risk for restenosis.

[0031] The data provided in the FIGS. (1-13) show that CMV virus infectssmooth muscle cells in vitro and that intermediate early expression ofviral protein can be seen (FIGS. 1-3). The affect of CMV infection insmooth muscle cells is shown affect actin reorganization as a market formigration activity (FIG. 4). Moreover, the infected cells were able tomigrate in a migration chamber, such as the one shown in FIG. 6. Thescheme shown in FIG. 5 provides that only infected cells have thecapability to migrate through a filter in a migration chamber. Thus,smooth muscle cells infected with CMV showed the ability to migrate,even to a much greater extent than non-infected smooth muscle cellstreated with the migration enhancing growth factor, PDGF (FIG. 7).

[0032] The next set of experiments were designed to determine whichprotein or proteins, encoded by the VMC genome, was responsible forconferring the migration activity on infected smooth muscle cells. Itwas first found that protein synthesis was required to confer themigration activity on infected smooth muscle cells (FIG. 9). Moreover,the suspect protein or proteins encoded by the CMV genome were not lategene production genes as evidenced by the fact that foscarnet (aninhibitor of HCMV late gene production) did not inhibit migration ofinfected smooth muscle cells (FIG. 9). This left four putative chemokinereceptors that are encoded by the CMV genome, US27, US28, UL33 and UL78(FIG. 10). In knock-out experiments, wherein each of the four foregoingchemokine receptor genes were knocked out, it was only a US28 knock outthat was able to inhibit smooth muscle cell migration activity whensmooth muscle cells were infected with the US28 knock out variety of CMV(FIG. 13). However, the ability of CMV infection to increase cellmigration of smooth muscle cells may not be only as US28 affect andthere are also ligand activity that needs to activate the US28 receptor.Moreover, CMV infection seems to also have an autocrine function inenhancing certain C-C chemokine production, such as RANTES (FIGS.11-12). Accordingly, the foregoing data provides the basis for thepresent invention.

[0033] Screening Assay

[0034] The present invention provides an assay for determiningtherapeutic activity of US28 receptor antagonists, comprising (a)obtaining and isolating smooth muscle cells into a first chamber of amigration device, wherein the first migration chamber comprises growthmedia chambers and is defined by a first side of a membrane and chamberwalls, and wherein the migration device comprises a second chamberdefined by the second side of the membrane and having an enclosed space;(b) infecting the smooth muscle cells with human cytomegalovirus (HCVM)containing a gene encoding the US28 receptor; (c) adding a candidatetherapeutic agent to the first chamber; and (d) determining the amountof cellular migration into the second chamber, whereby inhibition ofcellular migration of infected smooth muscle cells indicates therapeuticactivity. Preferably, the smooth muscle cells are isolated frompulmonary arteries. Preferably, the membrane has a pore size of fromabout 2 to about 10 microns. Most preferably, the membrane pore size isabout 3 microns. Preferably the amount of cellular migration isdetermined by an assay for counting the number of smooth muscle cells inthe second chamber wherein the assay for counting the number of smoothmuscle cells is selected from the group consisting of microscopic cellcounting per unit area, radiolabeling the smooth muscle cells andcounting radioactivity in the second chamber, attaching a fluorescentprobe to the smooth muscle cells and measuring fluorescence within thesecond chamber, and combinations thereof. Preferably, the infectedsmooth muscle cells are further stimulated with ligand to enhancemigration activity, wherein the ligand is a C-C chemokine. Preferably,the C-C ligand is selected from the group consisting of RANTES, MCP-1,MIP-1α, MIP-1β, and combinations thereof.

[0035] KHSV-encoded vMIP-2

[0036] KSHV-(Kaposi's sarcoma-associated herpes virus) encoded vMipalpha and beta has been described as having angiogenic and HIVinhibitory functions (Boshoff et al., Science 278:290-294, 1997). It hasalso been described as a broad-spectrum chemokine antagonist (Kledal etal., Science 277:1656-1659, 1997). The present invention adds to thetherapeutic uses for KHSV-encoded MIP for treating atherosclerosis,restenosis, chronic rejection syndrome and GVHD.

[0037] Fractalkine

[0038] Results from several studies showed that the polypeptidefractalkine is a ligand for US28 receptor and functions as a US28antagonist through competitive binding. Fractalkine has been describedin Kledal et al. FEBS Lett. 441:209-214, 1998. The present inventionadds to the therapeutic uses for fractalkine for treatingatherosclerosis, restenosis, chronic rejection syndrome and GVHD.

[0039] Herbimycin

[0040] Herbimycin A is a PTK (protein tyrosine kinase) pathwayinhibitor. It is available commercially (Sigma). In FIG. 15, the effectof herbimycin A on US28 SMC (smooth muscle cell) migration showedherbimycin A was effective in inhibiting US28 transfected smooth musclecell migration. Thus, it appears that US28 SMC migration is mediatedthrough a PTK pathway. The present invention adds to the therapeuticuses for herbimycin for treating atherosclerosis, restenosis, chronicrejection syndrome and GVHD.

[0041] Antisense

[0042] US28 is made off of two different transcripts, one only containsthe US28 ORF and the other contains US27/28 ORF's. Both use the samepoly-A signal. Antisense oligo sequences as US28 antagonists for bothUS27 and US28 are as follows:

[0043] US27-5′-1---ATT TGT AGA GGT GGT CAT [SEQ ID NO. 9]

[0044] US27-5′-2---GCT CAC CTG CGT TAA GGT [SEQ ID NO. 10]

[0045] US27-5′-3---GTG CTG TTT AAG GTG TGG [SEQ ID NO. 11]

[0046] US27-5′-4---AGT GTA CTC GAA CAA CTG [SEQ ID NO. 12]

[0047] US27-5′-5---CAA CCA TAC CCC GTT GGC [SEQ ID NO. 13]

[0048] US27-3′-1---TTC ACG CAG CAA CAG GCG [SEQ ID NO. 14]

[0049] US27-3′-2---CCT GGT AAG GTA TAT CCT [SEQ ID NO. 15]

[0050] US27-3′-3---GTA GCT CAA TAT CAA TGT [SEQ ID NO. 16]

[0051] US27-3′-4---GCC CTT CTT TGT ATG TCC [SEQ ID NO. 17]

[0052] US27-3′-5---ATG GGT ACG TTT GGT GTG [SEQ ID NO. 18]

[0053] US28-5′-1---CGT CGT CGT CGG TGT CAT [SEQ ID NO. 19]

[0054] US28-5′-2---CGT CGT GAG TTC CGC GGT [SEQ ID NO. 20]

[0055] US28-5′-3---CAG GGA GTC GCT TCA TCG [SEQ ID NO. 21]

[0056] US28-5′-4---TGA TTA AGC ACG TCG GTG [SEQ ID NO. 22]

[0057] US28-5′-5---GAA GAG AAA GAC AAC GCC [SEQ ID NO. 23]

[0058] US28-3′-1---GCT GTG GTA CCA GGA TAC [SEQ ID NO. 24]

[0059] US28-3′-2---CTC CGA CGC GAA AAG CTC [SEQ ID NO. 25]

[0060] US28-3′-3---GTC TCT CTT CGG CTC GGC [SEQ ID NO. 26]

[0061] US28-3′-4---CGG ACA GCG TGT CGG AAG [SEQ ID NO. 27]

[0062] US28-3′-5---GAG ACG CGA CAC GCC TCG [SEQ ID NO. 28]

[0063] Additional antisense sequences are provided as SEQ ID NOS 2-8.

[0064] Pharmaceutical Formulation

[0065] The inventive method in the form of a pharmaceutical compositioncomprising a US28 antagonist can be administered to a patient either byitself (complex or combination) or in pharmaceutical compositions whereit is mixed with suitable carriers and excipients. A US28 antagonist canbe administered parenterally, such as by intravenous injection orinfusion, intraperitoneal injection, subcutaneous injection, orintramuscular injection. A US28 antagonist can be administered orally orrectally through appropriate formulation with carriers and excipients toform tablets, pills, capsules, liquids, gels, syrups, slurries,suspensions and the like. A US28 antagonist can be administeredtopically, such as by skin patch, to achieve consistent systemic levelsof active agent. A US28 antagonist is formulated into topical creams,skin or mucosal patch, liquids or gels suitable to topical applicationto skin or mucosal membrane surfaces. A US28 antagonist can beadministered by inhaler to the respiratory tract for local or systemictreatment of HIV infection.

[0066] The dosage of the US28 antagonist suitable for use with thepresent invention can be determined by those skilled in the art fromthis disclosure. The US28 antagonist will contain an effective dosage(depending upon the route of administration and pharmacokinetics of theactive agent) of the US28 antagonist and suitable pharmaceuticalcarriers and excipients, which are suitable for the particular route ofadministration of the formulation (i.e., oral, parenteral, topical or byinhalation). The active US28 antagonist is mixed into the pharmaceuticalformulation by means of mixing, dissolving, granulating, dragee-making,emulsifying, encapsulating, entrapping or lyophilizing processes. Thepharmaceutical formulations for parenteral administration includeaqueous solutions of the active US28 antagonist in water-soluble form.Additionally, suspensions of the active US28 antagonist may be preparedas oily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil, or synthetic fatty acid ester,such as ethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances, which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. The suspension may optionally contain stabilizers or agents toincrease the solubility of the complex or combination to allow for moreconcentrated solutions.

[0067] Pharmaceutical formulations for oral administration can beobtained by combining the active compound with soild excipients, such assugars (e.g. lactose, sucrose, mannitol of sorbitol), cellulosepreparations (e.g., starch, methyl cellulose, hydroxypropylmethylcellulose, and sodium carboxymethyl cellulose), gelaten, gums, orpolyvinylpyrrolidone. In addition, a disintegrating agent may be added,and a stabilizer may be added.

1 28 1 1087 DNA Human cytomegalovirus 1 aaacgtcatc tcgccgacgt ggtgaaccgctcatatagac caaaccggac gctgcctcag 60 tctctcggtg cgtggaccag acggcgtccatgcaccgagg gcagaactgg tgctatcatg 120 acaccgacga cgacgaccgc ggaactcacgacggagtttg actacgatga agacgcgact 180 ccttgtgttt tcaccgacgt gcttaatcagtcaaagccag ttacgttgtt tctgtacggc 240 gttgtctttc tcttcggttc catcggcaacttcttggtga tcttcaccat cacctggcga 300 cgtcggattc aatgctccgg cgatgtttactttatcaacc tcgcggccgc cgatttgctt 360 ttcgtttgta cactacctct gtggatgcaatacctcctag atcacaactc cctagccagc 420 gtgccgtgta cgttactcac tgcctgtttctacgtggcta tgtttgccag tttgtgtttt 480 atcacggaga ttgcactcga tcgctactacgctattgttt acatgagata tcggcctgta 540 aaacaggcct gccttttcag tattttttggtggatctttg ccgtgatcat cgccattcca 600 cactttatgg tggtgaccaa aaaagacaatcaatgtatga ccgactacga ctacttagag 660 gtcagttacc cgatcatcct caacgtagaactcatgcttg gtgctttcgt gatcccgctc 720 agtgttatca gctactgcta ctaccgcatttccagaatcg ttgcggtgtc tcagtcgcgc 780 cacaaaggtc gcattgtacg ggtacttatagcggtcgtgc ttgtctttat catcttttgg 840 ctgccgtacc acctaacgct gtttgtggacacgttaaaac tcctcaaatg gatctccagc 900 agctgcgagt tcgaaagatc gctcaaacgtgcgctcatct tgaccgagtc gctcgccttt 960 tgtcactgtt gtctcaatcc gctgctgtacgtcttcgtgg gcaccaagtt tcggcaagaa 1020 ctacactgtc tgctggccga gtttcgccagcgactctttt cccgcgatgt atcctggtac 1080 cacagca 1087 2 20 DNA ArtificialSequence US28 receptor antisense receptor/specific antisense molecule 2ctggctttga ctgattaagc 20 3 20 DNA Artificial Sequence US28 receptorantisense receptor/specific antisense molecule 3 catgatagca ccagttctgc20 4 20 DNA Artificial Sequence US28 receptor antisensereceptor/specific antisense molecule 4 ccggagcatt gaatccgacg 20 5 20 DNAArtificial Sequence US28 receptor antisense receptor/specific antisensemolecule 5 gctggctagg gagttgtgat 20 6 20 DNA Artificial Sequence US28receptor antisense receptor/specific antisense molecule 6 ctggctttgactgattaagc 20 7 20 DNA Artificial Sequence US28 receptor antisensereceptor/specific antisense molecule 7 aaacaatagc gtagtagcga 20 8 20 DNAArtificial Sequence US28 receptor antisense receptor/specific antisensemolecule 8 ttggtcacca ccataaactg 20 9 18 DNA Artificial Sequence US27receptor antisense receptor/specific antisense molecule 9 atttgtagaggtggtcat 18 10 18 DNA Artificial Sequence US27 receptor antisensereceptor/specific antisense molecule 10 gctcacctgc gttaaggt 18 11 18 DNAArtificial Sequence US27 receptor antisense receptor/specific antisensemolecule 11 gtgctgttta aggtgtgg 18 12 18 DNA Artificial Sequence US27receptor antisense receptor/specific antisense molecule 12 agtgtactcgaacaactg 18 13 18 DNA Artificial Sequence US27 receptor antisensereceptor/specific antisense molecule 13 caaccatacc ccgttggc 18 14 18 DNAArtificial Sequence US27 receptor antisense receptor/specific antisensemolecule 14 ttcacgcagc aacaggcg 18 15 18 DNA Artificial Sequence US27receptor antisense receptor/specific antisense molecule 15 cctggtaaggtatatcct 18 16 18 DNA Artificial Sequence US27 receptor antisensereceptor/specific antisense molecule 16 gtagctcaat atcaatgt 18 17 18 DNAArtificial Sequence US27 receptor antisense receptor/specific antisensemolecule 17 gcccttcttt gtatgtcc 18 18 18 DNA Artificial Sequence US27receptor antisense receptor/specific antisense molecule 18 atgggtacgtttggtgtg 18 19 18 DNA Artificial Sequence US28 receptor antisensereceptor/specific antisense molecule 19 cgtcgtcgtc ggtgtcat 18 20 18 DNAArtificial Sequence US28 receptor antisense receptor/specific antisensemolecule 20 cgtcgtgagt tccgcggt 18 21 21 DNA Artificial Sequence US28receptor antisense receptor/specific antisense molecule 21 caaggagtcgcgtcttcatc g 21 22 18 DNA Artificial Sequence US28 receptor antisensereceptor/specific antisense molecule 22 tgattaagca cgtcggtg 18 23 18 DNAArtificial Sequence US28 receptor antisense receptor/specific antisensemolecule 23 gaagagaaag acaacgcc 18 24 18 DNA Artificial Sequence US28receptor antisense receptor/specific antisense molecule 24 gctgtggtaccaggatac 18 25 18 DNA Artificial Sequence US28 receptor antisensereceptor/specific antisense molecule 25 ctccgacgcg aaaagctc 18 26 18 DNAArtificial Sequence US28 receptor antisense receptor/specific antisensemolecule 26 gtctctcttc ggctcggc 18 27 18 DNA Artificial Sequence US28receptor antisense receptor/specific antisense molecule 27 cggacagcgtgtcggaag 18 28 18 DNA Artificial Sequence US28 receptor antisensereceptor/specific antisense molecule 28 gagacgcgac acgcctcg 18

We claim:
 1. An assay for determining therapeutic activity of US28receptor antagonists, comprising: (a) obtaining and isolating smoothmuscle cells into a first chamber of a migration device, wherein thefirst migration chamber comprises growth media chambers and is definedby a first side of a membrane and chamber walls, and wherein themigration device comprises a second chamber defined by the second sideof the membrane and having an enclosed space; (b) infecting the smoothmuscle cells with human cytomegalovirus (HCVM) containing a geneencoding the US28 receptor; (c) adding a candidate therapeutic agent tothe first chamber; and (d) determining the amount of cellular migrationinto the second chamber, whereby inhibition of cellular migration ofinfected smooth muscle cells indicates therapeutic activity.
 2. Theassay of claim 1 wherein the smooth muscle cells are isolated frompulmonary arteries.
 3. The assay of claim 1 wherein the membrane has apore size of from about 2 to about 10 microns.
 4. The assay of claim 3wherein the membrane pore size is about 3 microns.
 5. The assay of claim1 wherein the amount of cellular migration is determined by an assay forcounting the number of smooth muscle cells in the second chamber whereinthe assay for counting the number of smooth muscle cells is selectedfrom the group consisting of microscopic cell counting per unit area,radiolabeling the smooth muscle cells and counting radioactivity in thesecond chamber, attaching a fluorescent probe to the smooth muscle cellsand measuring fluorescence within the second chamber, and combinationsthereof.
 6. A method for treating atherosclerosis, restenosis, chronicrejection syndrome and graft versus host disease (GVHD), comprisingadministering an effective amount of an agent that is a US28 receptorantagonist, wherein a US28 receptor antagonist comprises an inhibitorcompound that prevents transduction of US28 receptor signal stimulatedby a US28 receptor ligand, wherein a US28 receptor ligand is selectedfrom the group consisting of RANTES, MIP-1α and MCP.
 7. The method ofclaim 6 wherein the US28 receptor antagonist is selected from the groupconsisting of an antibody that binds to an extracellular portion of theUS28 receptor, an antisense oligonucleotide having a nucleic acidsequence antisense to the US28 cDNA and inhibiting translation of US28expression in infected smooth muscle cells, and US28 receptor antagonistis selected from the group consisting of an antibody that binds to anextracellular portion of the US28 receptor, and a US28 bindingantagonist, wherein the US28 binding antagonist is selected from thegroup consisting of KHSV encoded vMIP-2, fractalkine, and herbimycin. 8.The method of claim 6 wherein the monoclonal antibody is chimeric orhumanized by means for humanizing non-human antibodies.
 9. The method ofclaim 6 wherein the US28 antisense sequences are selected from the groupconsisting of SEQ ID NOS. 2-28.
 10. A method for enhancing cellularmigration, comprising infecting a cell with a viral nucleic acidcontaining a gene encoding CVM US28 receptor or tansfecting a cell witha vector comprising the cDNA sequence for US28 operably linked to aviral promoter sequence, and stimulating the transfected or infectedcell with a US28 receptor ligand, selected from the group consisting ofRANTES, MIP-1α and MCP1.